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All tissues undergo continuous reconditioning via the complex orchestration of changes in tissue protein synthesis and breakdown rates. Skeletal muscle tissue has been well studied in this regard, and has been shown to turnover at a rate of 1-2% per day in vivo in humans. Few data are available on protein synthesis rates of other tissues. Because of obvious limitations with regard to brain tissue sampling no study has ever measured brain protein synthesis rates in vivo in humans. Here, we applied stable isotope methodology to directly assess protein synthesis rates in neocortex and hippocampus tissue of six patients undergoing temporal lobectomy for drug-resistant temporal lobe epilepsy (Clinical trial registration: NTR5147). Protein synthesis rates of neocortex and hippocampus tissue averaged 0.17 ± 0.01 and 0.13 ± 0.01%/h, respectively. Brain tissue protein synthesis rates were 3-4-fold higher than skeletal muscle tissue protein synthesis rates (0.05 ± 0.01%/h; P < 0.001). In conclusion, the protein turnover rate of the human brain is much higher than previously assumed.

Obesity and its associated complications have reached epidemic proportions in Western-type societies. Concomitantly, the obesity incidence in developing countries is increasing. One hallmark of obesity is the differentiation of pre-adipocytes into mature triglyceride-loaded adipocytes present in subcutaneous and visceral adipose tissue depots. This may ultimately lead to dysfunctional adipose tissue together with detrimental changes in the profiles of (pre-)adipocyte-secreted proteins, known as adipokines. Obesity-induced alterations in adipokine profiles contribute to the development of obesity-associated disorders. Consequently, the interest in the molecular events responsible for adipose tissue modifications during weight gain and weight loss as well as in the aetiology of obesity-associated disorders is growing. Molecular mechanisms involved in pre-adipocyte differentiation and alterations in adipokine profiles have been examined at the gene and protein level by high-throughput technologies. Independent proteomics studies have contributed significantly to further insight into adipocyte biology, particularly with respect to adipokine profiling. In this review novel findings obtained with adipo-proteomics studies are highlighted and the relevance of proteomics technologies to further understand molecular aspects of adipocyte biology is discussed.

Background: The influence of single nucleotide polymorphisms (SNP) of the FTO gene has been shown to change over time., Objective: The aim was to investigate the relationship between a SNP of the FTO gene (rs9939609) and obesity-related characteristics longitudinally during childhood and puberty., Design: From 101 children (58 boys and 43 girls), the FTO (rs9939609) genotype and yearly anthropometric data from birth to age 7 yr were determined. From ages 12 to 17 yr, we measured anthropometry, body composition, leptin concentrations, physical activity, hours watching television, and attitude toward eating yearly; parental characteristics were determined as well., Results: At age 17 yr, 20% of the children were overweight/obese, and 88% of the overweight/obese children had the A allele in contrast to 45% of the lean children (P < 0.001). The A allele carriers had a higher fat mass index (kilograms per square meter) and higher leptin concentrations (micrograms per liter) during puberty, except at age 14 yr. Multiple regression analyses with body mass index (BMI; kilograms per square meter) as the dependent variable showed that at ages 12 and 17 yr, dietary restraint score, disinhibition score, BMI of the mother, and the FTO A allele significantly contributed to the model (R(2) = 0.29, P < 0.002; and R(2) = 0.39, P < 0.001). At age 14 yr, dietary restraint score, disinhibition score, and leptin concentrations per kilogram of fat mass significantly contributed to the model (R(2) = 0.25; P < 0.02)., Conclusion: The FTO A allele (rs9939609) is associated with higher BMI, fat mass index, and leptin concentrations from the age of 12 yr, whereas the associations show a dip at ages 13-14 yr and become stronger at age 17 yr. The dip is presumably caused by the dominating endocrinological changes at midpuberty.

Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery.

Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.

Background: Acute psychological stress is associated with eating in the absence of hunger., Objective: To investigate if BclI and FTO polymorphisms are associated with eating in the absence of hunger as a result of acute psychological stress., Methods: FTO (rs9939609) and BclI were genotyped in 98 subjects (BMI=23.9+/-3.3kg/m(2)). In a randomized crossover design, the 'eating in absence of hunger' protocol was measured as a function of acute stress vs. a control task and of STAI (State Trait Anxiety Index) state scores., Results: In comparison with the FTO T allele, the A allele was associated with an increased feelings of hunger after food intake in the stress (11+/-10 vs. 18+/-15, p<0.01) and control condition (12+/-9 vs. 16+/-12, p<0.05), even though food intake was not different. For the first time, it was observed that in comparison to the BclI C/C genotype, the BclI G/G genotype was associated with higher STAI states scores at 0, 10, and 20min after the stress condition (30.8+/-6.4 vs. 36.3+/-8.2; 28.3+/-5.5 vs. 32.3+/-7.5; 27.7+/-6.1 vs. 31.2+/-7.5, p<0.05). Additionally, the BclI G/G genotype was associated with a larger difference in energy intake between the stress and control condition, in comparison with the BclI C/C genotype (136.6+/-220.4 vs. 29.4+/-176.3kJ, p<0.04)., Conclusion: In concordance with previous studies, the FTO A allele is related to a lower feeling of hunger after a standardized meal. For the first time, the BclI G/G genotype is shown to be associated with increased sensitivity to psychological stress, and increased eating in the absence of hunger after stress., Practice Implications: Interventions to reduce body weight should consider the subjects' genetic background., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

We have applied our recently designed proteomics apparoach to search for protein changes in the in vivo adipocyte-enriched proteome from 8 overweight/obese subjects who underwent an intervention of 5 weeks of a very low calorie diet followed by 3 weeks of a normal diet. On average, persons lost 9.5 kg body weight largely contributed by the loss of fat mass (7.1 kg). Various parameters of adiposity and lipid metabolism changed significantly. Proteomics analysis revealed 6 significantly changed proteins. Analysis indicates that fructose-bisphosphate aldolase C and tubulin beta 5 are potential biomarkers for the present intervention. Further, identified proteins indicate a reduced intracellular scaffolding of GLUT4 (ALDOC, TUBB5, ANXA2), an increased uptake of fatty acids (FABP4), an improved inflammatory profile of the adipose tissue (ApoA1, AOP1) and a change in fat droplet organization (vimentin). Correlation analysis between changes in protein spot intensities and parameters of adiposity or lipid metabolism points to a link between aldo-ketoreductase 1C2 and parameters of adiposity, between FABP4 and parameters of lipid metabolism, and between proteins for beta-oxidation (HADH, ACADS, ACAT1) and FFA levels. Altogether, our findings underscore the potential value of in vivo proteomics for human intervention studies.

Background: The common rs9939609 single nucleotide polymorphism (SNP) in the fat mass and obesity-associated (FTO) gene is associated with adiposity, possibly by affecting satiety responsiveness., Objective: The objective was to determine whether postprandial responses in hunger and satiety are associated with rs9939609, taking interactions with other relevant candidate genes into account., Design: Sixty-two women and 41 men [age: 31 +/- 14 y; body mass index (in kg/m(2)): 25.0 +/- 3.1] were genotyped for 5 SNPs in FTO, DNMT1, DNMT3B, LEP, and LEPR. Individuals received fixed meals provided in energy balance. Hunger and satiety were determined pre- and postprandially by using visual analog scales., Results: A general association test showed a significant association between postprandial responses in hunger and satiety with rs9939609 (P = 0.036 and P = 0.050, respectively). Individuals with low postprandial responses in hunger and satiety were overrepresented among TA/AA carriers in rs9939609 (FTO) compared with TT carriers (dominant and additive model: P = 0.013 and P = 0.020, respectively). Moreover, multifactor dimensionality reduction showed significant epistatic interactions for the postprandial decrease in hunger involving rs9939609 (FTO), rs992472 (DNMT3B), and rs1137101 (LEPR). Individuals with a low postprandial decrease in hunger were overrepresented among TA/AA (dominant), CC/CA (recessive), and AG/GG (dominant) carriers in rs9939609 (FTO), rs992472 (DNMT3B), and rs1137101 (LEPR), respectively (n = 39), compared with TT, AA, and/or AA carriers in these SNPs, respectively (P = 0.00001). Each SNP had an additional effect., Conclusions: Our results confirm a role for FTO in responsiveness to hunger and satiety cues in adults in an experimental setting. The epistatic interaction suggests that DNA methylation, an epigenetic process, affects appetite.

The specificity of proteins identified by proteomics as biomarkers for defined conditions or as components of biological processes and pathways is crucial. We critically reviewed differentially expressed proteins from comparative proteomic studies identified by 2-DE followed by MS, especially with MALDI technique. Based on 66 of those studies, a list of 44 proteins is presented as generally detected proteins regardless of species, in vivo or in vitro conditions, tissues and organs, and experimental objective. Similarly, a list of 28 generally detected protein families is presented. The enriched functions linked to these generally detected proteins reveal that there are some common biological features beyond the technical limitations. Cellular stress response can be the universal reason as to why these proteins are generally expressed differentially. Using those proteins as biomarkers for cellular processes other than stress response should be done with caution. In future proteomic studies more profound approaches should be applied to look beyond these proteins to find specific biomarkers. Our results are discussed in relation to a recent viewpoint publication by Petrak et al. [Proteomics 2008, 8, 1744-1749].

Background: In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls., Results: Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed., Conclusion: Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of in vitro obtained findings when translating them to the in vivo situation.

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition.

It is suggested that colorectal cancer might be prevented by changes in diet, and vegetable consumption has been demonstrated to have a protective effect. Until now, little is known about the effects of vegetable consumption at the proteome level. Therefore, the effect of increased vegetable intake on the protein expression in the colonic mucosa of healthy mice was studied. Aim was to identify the proteins that are differentially expressed by increased vegetable consumption and to discriminate their possible role in the protection against colorectal cancer. Mice were fed four different vegetable diets, which was followed by analysis of total cellular protein from colonic mucosal cells by a combination of 2-DE and MS. We found 30 proteins that were differentially expressed in one or more diets as compared to the control diet. Six could be identified by MALDI-TOF MS: myosin regulatory light chain 2, carbonic anhydrase I, high-mobility group protein 1, pancreatitis-associated protein 3, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase oligomycin sensitivity conferral protein. Alterations in the levels of these proteins agree with a role in the protection against colon cancer. We conclude that these proteins are suitable markers for the health effect of food on cancer. The observed altered protein levels therefore provide support for the protective effects of vegetables against colorectal cancer.